how reliable are the measurements of residual gluten in gluten-free foods?
DESCRIPTION
International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012TRANSCRIPT
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How reliable are the measurements of residual gluten in
gluten-free foods?
Päivi Kanerva
Department of Food and Environmental Sciences
University of Helsinki, Finland
11th International Gluten Workshop Beijing, China
August 12-15, 2012
www.helsinki.fi/yliopisto
Introduction
Conditions that affect reliability of gluten measurements:
• Variety of gluten-detecting antibodies
• Different reference materials
• Protein hydrolysis
• Protein deamidation
Conclusions
Content
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
www.helsinki.fi/yliopisto
• Coeliac disease is one of the most common food intolerances in the world, with prevalence of 1-2 %
• Gluten proteins are known to be an environmental factor causing coeliac disease
• Gluten proteins of wheat and similar proteins of barley and rye are all harmful for coeliacs
• Harmful proteins trigger an adverse immunoreaction in people with coeliac disease leading to destruction of small intestine villi
• Current treatment for coeliac disease is a gluten-free diet
Introduction Coeliac disease
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
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Introduction Measuring gluten content
• Gluten content is measured by immunological ELISA methods.
• Methods are based on prolamin-recognizing antibodies.
• The method currently recommended by Codex is known by names R5-ELISA or the method of Mendez
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
Sandwich Competitive
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Variety of gluten-detecting antibodies
R5 antibody raised against rye secalin (Sorell et al. 1998)
– currently in use
Recognizes prolamins of wheat, barley and rye; reacts mainly with QQPFP
ω-gliadin antibody raised against wheat gliadin (Skerritt&Hill 1990)
– predecessor of R5
Recognizes heat-stable omega-type prolamins
α-gliadin antibody raised against wheat gliadin (Spaenij-Dekking et al. 2004)
Recognizes T-cell stimulatory epitopes of wheat, barley and rye
G12 and A1 antibodies raised against 33mer (Moron et al. 2008)
Recognizes prolamins of wheat, barley and rye, and weak reaction to oat prolamins; G12 reacts mainly with QPQLPY, A1 with QLPYPQP
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
Kanerva et al 2011 Agric Food Sci 20:206-216.
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• Reference material has a major influence on the quantification.
• Reference material should be similar to the analysed protein; however, complexicity of prolamins makes it difficult
• Different standards are used today in gluten analysis:
PWG gliadin other gliadin standards (e.g. Sigma) Peptide standards (synthetic or hydrolyzed)
Different reference materials
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
Measurement of gluten with R5 ELISA using different reference materials
Amount of barley flour (%) Amount of barley flour (%)
Glu
ten
co
nte
nt
(pp
m)
Ho
rde
in c
on
ten
t (p
pm
)
Gliadin standard Hordein standard
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• Hydrolysis of proteins occurs during many food manufacturing processes, e.g. brewing
• Hydrolysis can be enhanced by enzymes: either added or endogenous
• High amounts of prolamin degrading enzymes is developed during grain germination
Hydrolysis of proteins
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
•Rye protein hydrolysis during sourdough
process
Sourdough from germinated rye contained
only 240-480 ppm of prolamins, which is
about 0,5% from original amount
Both sandwich and competitive assay gave
similar results
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Protein deamidation
In addition to hydrolysis, solubility of gluten can be improved by deamidation
Deamidation improves also foam and emulsion forming cababilities of gluten proteins
Deamidation can be done by acid or base treatment or enzymatically
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
Deamidation is a conversion of glutamine or asparagine to glutamic acid or aspartic acid
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
Chemical deamidation of vital gluten by acid treatment
Deamidation of gluten significantly
reduced its immunological detection
by the prolamin specific antibodies
R5 and omega-gliadin.
R5 sandwich ELISA
x 600
R5 competitive ELISA
x 125
Omega-gliadin sandwich
ELISA
Kanerva et al. 2011. J Cereal Sci
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Reliability of the measurements of gluten content is questioned if the complexity of prolamins and changes in their structures are not carefully considered.
reliability can be substantially improved by selecting right reference material
reactivity of different prolamins with different antibodies should be taken into account and results interpreted accordingly
consequences of modification to protein reactivity with antibodies should be evaluated
Conclusions
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva
Thank you Cereal Technology Group at the University of Helsinki
Professor
Hannu Salovaara
University lecturer
Tuula Sontag-Strohm
Researchers
Päivi Kanerva
Xin Huang
Technician
Outi Brinck